1. Volume 15, Issue 5, Pages 574-588 (November 2014)
Transcriptional Pause Release Is a Rate-Limiting Step for Somatic Cell Reprogramming 
Longqi Liu, Yan Xu, Minghui He, Meng Zhang, Fenggong Cui, Leina Lu, Mingze Yao, Weihua Tian, Christina Benda, Qiang Zhuang, Zhijian Huang, Wenjuan Li, Xiangchun Li, Ping Zhao, Wenxia Fan, Zhiwei Luo, Yuan Li, Yasong Wu, Andrew P. Hutchins, Dongye Wang, Hung-Fat Tse, Axel Schambach, Jon Frampton, Baoming Qin, Xichen Bao, Hongjie Yao, Biliang Zhang, Hao Sun, Duanqing Pei, Huating Wang, Jun Wang, Miguel A. Esteban 
Cell Stem Cell 
Volume 15, Issue 5, Pages (November 2014)
DOI: /j.stem
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2. Cell Stem Cell 2014 15, 574-588DOI: (10.1016/j.stem.2014.09.018)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1 Pol II Is Paused at Pluripotency Genes in Reprogramming
(A) GRO-sequencing maps of Rcor1 and Pttg1 in ESCs from the reanalysis of a previous data set (GSE27037).
(B) Venn diagrams show the number of genes upregulated in the first and second wave of reprogramming that belong to class II in MEFs, ESCs, or both.
(C) Schematic representation describing how to calculate the TR.
(D) Percentage of second wave genes that experience Pol II pausing at day 8 of reprogramming. D indicates day.
(E) Pol II TR distribution of second wave genes paused at day 8 of reprogramming compared to day 5 and untransduced MEFs.
(F) Box plots showing the average TR of second wave genes paused at day 8 of reprogramming compared to day 5 and untransduced MEFs.
(G) Percentage of second wave genes paused at day 8 of reprogramming that experience pause release in ESCs.
(H) Pol II TR distribution of second wave genes paused at day 8 of reprogramming that are released in ESCs.
(I) Box plots showing the average TR of second wave genes paused at day 8 of reprogramming that are released in ESCs.
(J and K) Pol II occupancy on Oct4 and Thy1 in MEFs, in ESCs, and at two different time points during reprogramming.
See also Figure S1.
Cell Stem Cell  , DOI: ( /j.stem )
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4. Figure 2
P-TEFb Regulates the Activation of Pluripotency Genes in the Late Phase of Reprogramming
(A) Western blot for endogenous CDK9 in MEFs and ESCs (left) or during reprogramming (right). Histone H3 (H3) was used as loading control.
(B) Phase contrast (left), fluorescence photographs (middle), and AP-stained wells (right) of MEFs reprogrammed with OSKM and CDK9-DN at day 10. An empty vector was used as a control (Ctrl; also hereafter in similar experiments). Scale bar represents 50 μm.
(C) Number of GFP+ colonies at day 12 in MEFs reprogrammed as in (B). Average colony numbers of three independent experiments are shown.
(D) Phase contrast (left), fluorescence photographs (middle), and AP-stained wells (right) of MEFs reprogrammed with OSKM and two independent shRNAs for Cdk9 (shCdk9) at day 10. shRNA against firefly luciferase was used as a control (shCtrl; also hereafter in similar experiments). Scale bar represents 50 μm.
(E) Number of GFP+ colonies at day 12 in MEFs reprogrammed as in (D). Average colony numbers of three independent experiments are shown.
(F and G) Number of GFP+ colonies at day 11 in MEFs reprogrammed with OSKM and an inducible CDK9-DN (teton CDK9-DN). Doxycycline (dox; 2 μg/ml) was administered daily for the indicated time periods. The dotted lines are used to highlight deviations from the mean number of colonies in untreated control wells (also hereafter in selected experiments). Average colony numbers of two independent experiments are shown.
(H) Heatmap of RNA-sequencing data showing the expression level of genes corresponding to the first (for days 3, 4, and 5) and second (for day 10) wave of reprogramming in MEFs reprogrammed with OSKM and empty vector or CDK9-DN.
(I) Number of RefSeq genes significantly differentially regulated (fold change >1.25, false discovery rate or FDR < 0.01) by CDK9-DN in the early phase (in all three time points) and late phase of OSKM reprogramming.
(J) Percentage of RefSeq and second wave genes upregulated and downregulated (fold change >1.25, FDR < 0.01) or unchanged by CDK9-DN at day 10 of reprogramming.
(K) qPCR for the indicated genes using RNA lysates from MEFs reprogrammed with OSKM and empty vector or CDK9-DN at day 9. Endo- indicates endogenous. Values are referred to untransduced MEFs.
The data in (C), (E), (F), (G), and (K) are reported as mean values ± standard deviation (SD) with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005). See also Figure S2 and Table S1.
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5. Figure 3 BRD4 Enhances Reprogramming
(A) Western blot for endogenous BRD4 in MEFs and ESCs (top) or during reprogramming (bottom). Histone H3 (H3) was used as the loading control.
(B) Phase contrast (left), fluorescence photographs (middle), and AP-stained wells (right) of MEFs reprogrammed with OSKM and shRNA control or two shRNAs for Brd4 (shBrd4) at day 10. Scale bar represents 50 μm.
(C) Number of GFP+ colonies at day 12 in MEFs reprogrammed as in (B). Average colony numbers of three independent experiments are shown.
(D and E) Number of GFP+ colonies in MEFs reprogrammed with OSKM and the BRD4 inhibitor JQ1 (100 nM). DMSO was used as a control. Colonies were counted at day 13 (D) or day 11 (E). Average colony numbers of two independent experiments are shown.
(F) Number of GFP+ colonies at day 11 in MEFs reprogrammed with OSKM and empty vector or BRD4. Average colony numbers of three independent experiments are shown.
(G) AP-stained wells of a representative experiment as in (F) at day 10.
(H) Schematic representation of the time of GFP+ colony appearance during OSKM reprogramming with or without exogenous BRD4.
(I) Immunofluorescence, phase contrast photographs, and chimeric mice with germline transmission (red arrow) obtained with a representative iPSC colony produced with OSKM and BRD4. Scale bar represents 50 μm.
The data in (C)–(F) are reported as mean values ± SD with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).
See also Figure S3.
Cell Stem Cell  , DOI: ( /j.stem )
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6. Figure 4 BRD4 Enhances Reprogramming through P-TEFb
(A) Schematic depiction of wild-type BRD4 and the different truncated BRD4 mutants used in this study.
(B) Immunoprecipitation (IP) of the different BRD4 variants (FLAG-tagged) shown in (A) in HEK293T cells. FLAG-tagged GFP was used as control. Membranes were immunoblotted (IB) for CDK9 and FLAG. Black arrows indicate specific bands.
(C) Number of GFP+ colonies at day 11 in MEFs reprogrammed with OSKM and empty vector or the indicated BRD4 variants. Average colony numbers of three independent experiments are shown.
(D) Schematic depiction of the location of the CDK9 binding site on BRD4 and the corresponding modifications performed to abolish their interaction.
(E) Immunoprecipitation of the indicated BRD4 variants (FLAG-tagged) in HEK293T cells. FLAG-tagged GFP was used as a control. Membranes were immunoblotted for CDK9 and FLAG.
(F and G) Number of GFP+ colonies at day 11 in MEFs reprogrammed with OSKM and empty vector or the indicated BRD4 variants. Average colony numbers of three (F) or two (G) independent experiments are shown.
(H) Number of human ESC-like colonies at day 28 in human urinary cells (hUCs) from three different donors reprogrammed with OSKM and BRD4-CTD.
(I) Phase contrast and immunofluorescence photographs of human iPSCs produced from urinary cells transduced with OSKM and BRD4-CTD. Scale bar represents 50 μm.
The data in (C) and (F)–(H) are reported as mean values ± SD with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).
See also Figure S4.
Cell Stem Cell  , DOI: ( /j.stem )
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7. Figure 5
BRD4 Regulates Transcriptional Pause Release at Pluripotency Genes
(A and B) qPCR for the indicated genes using RNA lysates from MEFs reprogrammed with OSKM and empty vector or BRD4-CTD at day 9 (A) and day 5 (B). Values are referred to untransduced MEFs.
(C) Upper: schematic representation of the Nanog, Oct4, Sall4, and Sox2 loci. The amplified regions are shown below. Lower: ChIP-qPCR of Pol II (total and Ser2P) binding at these loci in MEFs reprogrammed with OSKM and empty vector or BRD4-CTD.
(D) AP staining of ESCs transduced with shRNA control or two shRNAs for Brd4. Scale bar represents 50 μm.
(E) qPCR for the indicated pluripotency genes using lysates from ESCs transduced with shRNA control or two shRNAs for Brd4.
(F) BRD4 occupancy on Nanog and Oct4 from the reanalysis of a previous data set (GSE36561).
The data in (A)–(C) and (E) are reported as mean values ± SD with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).
See also Figure S5.
Cell Stem Cell  , DOI: ( /j.stem )
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8. Figure 6 HEXIM1 Reduces P-TEFb Activity and Blocks Reprogramming
(A) Immunoprecipitation of CDK9 using lysates from MEFs reprogrammed with OSKM and empty vector or HEXIM1 at day 9. Membranes were immunoblotted for HEXIM1, BRD4, and CDK9.
(B) Phase contrast (left), fluorescence photographs (middle), and AP-stained wells (right) of MEFs reprogrammed with OSKM and HEXIM1 at day 10. Scale bar represents 50 μm.
(C) Number of GFP+ colonies at day 13 in MEFs reprogrammed as in (B). Average colony numbers of three independent experiments are shown.
(D) Immunoprecipitation of CDK9 using lysates from MEFs reprogrammed with OSKM and shRNA control or two shRNAs for Hexim1 (shHex) at day 9. Membranes were immunoblotted for HEXIM1, BRD4, and CDK9.
(E) AP-stained wells of MEFs reprogrammed with OSKM and shRNA control or two shRNAs for Hexim1 at day 10.
(F) Number of GFP+ colonies at day 11 in MEFs reprogrammed as in (E). Average colony numbers of two independent experiments are shown.
(G) Phase contrast (left) and fluorescence photographs (right) of MEFs reprogrammed with OSKM and shRNA control or shRNA for Hexim1 in iSF1 medium at day 14. Scale bar represents 50 μm.
(H) OCT4-GFP+ cells analyzed by FACS in MEFs reprogrammed as in (G) at day 14.
(I and J) qPCR for the indicated genes using RNA lysates from MEFs reprogrammed with OSKM and empty vector or HEXIM1 (I) and shRNA control or shRNA for Hexim1 (J) at day 9. Values are referred to untransduced MEFs.
The data in (C), (F), (I), and (J) are reported as mean values ± SD with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).
See also Figure S6.
Cell Stem Cell  , DOI: ( /j.stem )
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9. Figure 7
KLF4 Interacts with CDK9 and Shares Overlapping Targets throughout the Genome
(A and B) Number of GFP+ colonies at day 12 in MEFs reprogrammed with OSK and the indicated vectors. Average colony numbers of three (A) or two (B) independent experiments are shown.
(C) Immunoprecipitation of the indicated proteins (FLAG-tagged) in HEK293T cells. FLAG-tagged GFP was used as control. Membranes were immunoblotted for CDK9 and FLAG.
(D) Coimmunoprecipitation of endogenous CDK9 with KLF4 in MEFs reprogrammed with OSKM at day 10. Antibodies against IgG were used as a control.
(E) Hierarchical clustering of the indicated transcription factors and chromatin regulators from the reanalysis of previous data sets (GSE11431 and GSE44286). Colors in the heatmap indicate the correlation between the ChIP-sequencing data sets.
(F and G) Distribution of CDK9, KLF4, and c-MYC ChIP-sequencing tags relative to the transcription start site (TSS) of RefSeq genes (F) or second wave genes (G) obtained from the reanalysis of previous data sets (GSE11431 and GSE44286).
(H) CDK9, KLF4, and c-MYC occupancy on Nanog obtained from the reanalysis of previous data sets (GSE11431 and GSE44286).
(I and J) Sequential ChIP of KLF4 and then CDK9 at the indicated gene promoters in ESCs (I) and MEFs reprogrammed with OSKM at two different time points (J). Untransduced MEFs were used as a control in (J).
(K) ChIP-qPCR for CDK9 on the Nanog and Oct4 promoters 48 hr after ESCs were transduced with shRNA control or shRNA for Klf4 (shKlf4).
(L) Upper: schematic representation of the Nanog and Oct4 loci and the regions amplified in the ChIP-qPCR. Lower: ChIP-qPCR of Pol II (total and Ser2P) binding to these loci 48 hr after transducing ESCs with shRNA control or shRNA for Klf4.
(M) Schematic representation of the main message of our work.
The data in (A), (B), and (I)–(L) are reported as mean values ± SD with the indicated significance (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005).
See also Figure S7.
Cell Stem Cell  , DOI: ( /j.stem )
Copyright © 2014 Elsevier Inc. Terms and Conditions